Toxicological research uses different analytical techniques for the quantification of compounds of interest which are detrimental to the organism. These techniques become even more relevant when detecting the use of illicit substances is needed, or detecting drugs, toxins or pollutants in biological samples.
Glucuronidation of chemical compounds is an essential part of the metabolism of toxic substances to facilitate their excretion of the organism. During this process, biochemical reactions occur which eventually give rise to glucuronide compounds, i.e., metabolites to which glucuronic acid has been conjugated by means of a covalent bond. This biological process has been extensively documented, as shown in Rowland, A. et al. (2013), The UDP-glucuronosyltransferases: their role in drug metabolism and detoxification, The International Journal of Biochemistry & Cell Biology, 45 (6), 1121-1132.
For the identification and quantification of glucuronide metabolites in biological fluids, enzymatic hydrolysis methods are used which consist of the use of glucuronidases from different origins. As an example, US patent application 20160076075 A1 discloses a method of enzymatic hydrolysis of glucuronide metabolites.
As a product of the reaction catalyzed by these enzymes, the glucuronide metabolites are degraded to their corresponding aglycones and glucuronic acid, which are subsequently detected by analytical techniques, as shown in Fox, E. et al. (2006), Quantitative analysis of buprenorphine and norbuprenorphine in urine using liquid chromatography tandem mass spectrometry, Journal of Analytical Toxicology, 30 (4), 238-244). The glucuronidase enzymes used for these purposes can be obtained from various organisms, such as Escherichia coli, Patella vulgata, Helix pomatia, Haliotis rufescens and from bovine liver (Kemp, P M and Cliburn, K D (2015), Comparison of Species-Specific β-Glucuronidase Hydrolysis of Cannabinoid Metabolites in Human Urine (No. DOT/FAA/PM-15/6)). Recently, a β-glucuronidase derived from Brachyspira pilosicoli bacterium has been reported (patent application GB1614546.8 (2016), Kura Biotec).
Once the biological sample containing the glucuronide metabolites is enzymatically hydrolyzed, there are different analytical techniques for the detection of the products of said hydrolysis, in which liquid chromatography coupled to mass spectrometry (LC-MS/MS as the initials of Liquid Chromatography-Mass Spectrometry) is one of the most commonly used. However, these applications generally use stages of sample preparation based on liquid-liquid extraction (LLE, as the initials of Liquid-Liquid Extraction; or SLE, for Supported Liquid Extraction) or solid phase extraction (SPE as the initials of Solid Phase Extraction, DPX (Disposable Pipette tip Extraction), as described in PCT patent application WO2013123253) as pre-chromatographic analysis, to clean impurities from the sample in order to prevent contaminants produced by metabolism that may interfere or affect the subsequent analysis. Although improvements have been made to facilitate the detection process of the compounds of interest, by eliminating the pre-analysis extraction step, such as the dilution and injection method (DS, dilute and Shoot LC-MS) (Deventer, K. et al. (2014), Dilute-and-shoot-liquid chromatography-mass spectrometry for urine analysis in doping control and analytical toxicology, Trac Trends in Analytical Chemistry, 55, 1-13) or removing the enzyme added in the hydrolysis step (β-Gone™, Phenomenex), these techniques generate loss of sensitivity in quantitation due to high dilution and retard the process of analysis, since an additional step must be considered.
The implementation of enzymes on an industrial scale previously requires the improvement of the catalytic properties of the same, since the majority of the enzymes are not stable in the working conditions. When enzymes are soluble in aqueous solutions, their separation from the substrates and products is difficult, which therefore limits their reutilization. In particular, for the purpose of drug analysis in biological samples, the enzymes used in the hydrolysis of the glucuronide metabolites, when found free in solution, obstruct the analysis columns and decrease the concentration of the enzyme available for the reaction, interfering in the final step of quantification. Moreover, multimeric enzymes can be dissociated in their subunits, depending on the dilution and pH when soluble, as it is the case of β-glucuronidase enzymes.
Due to the above, the soluble enzymes must be immobilized to allow their use for prolonged periods. In this way, the immobilization of enzymes is a process in which they are fixed in a support to give rise to insoluble forms able to maintain their catalytic activity and to increase their stability (Arroyo, M. (1998), Inmovilización de enzimas. Fundamentos, métodos y aplicaciones, Ars Pharvaceutica, 39 (2), 23-39). Although immobilization of enzymes is generally considered to improve their stability in general, this does not always occur if the immobilization has not been properly designed, and it may even be decreased if the support produces undesired interactions with the enzyme (Mateo, C. et al. (2007), Improvement of enzyme activity, stability and selectivity via immobilization techniques, Enzyme and microbial Technology, 40 (6), 1451-1463).
Regarding the immobilization of enzymes, the book of Guisan, J. M. (2006. Immobilization of enzymes and cells. Second Edition. Methods in Biotechnology; 22. Humana Press. 465 p. Chapter 1: Immobilization of enzymes as the 21st Century begins: an already solved problem or still an exciting challenge?) suggests that many companies can produce enzymes, however they are not capable of producing them in supports because the development of protocols for the immobilization of enzymes is critical. It also indicates that enzymes possess amino acid residues that are capable of interacting, either by adsorption or covalently, with the immobilization supports by means of different mechanisms and, therefore, protocols are required that are efficient for each enzyme in terms of its activity, stability, selectivity and absence of inhibitors.
The state of the art is broad in relation to enzyme immobilization techniques in general. The publication Brady, D. and Jordaan, J. (2009. Advances in enzyme immobilisation. Biotechnology Letters, 31 (11), 1639) describes supports for immobilization of enzymes, wherein commercial resins are disclosed and microcapsules, dendrispheres or spherenzymes, among others.
As is well known in the field of biochemistry, the structure of an enzyme, whether its composition of amino acid residues and the three-dimensional conformation of these differ from one to another. The publication of Singh, R. K. et al (2013), From engineering to protein immobilization. Promising strategies for the upgrade of Industrial enzymes, International Journal of Molecular Sciences, 14 (1), 1232-1277, describes that the content of lysine residues affects the binding of an enzyme to glutaraldehyde-agarose resins and that, therefore, variations in lysine content may produce differences in enzyme immobilization since conformational changes can occur, altering the affinity for the substrate.
PCT application WO 03/076640 describes the immobilization of the enzyme D-amino acid oxidase (DAAO) originated from Rhodotorula dracilis or Trigonopsis variabilis on agarose modified with glyoxyl groups, wherein it was observed that the R. dracilis enzyme was considerably more stabilized than the enzyme originated from T. variabilis, indicating that the same immobilization protocol does not necessarily work for the same enzyme of any origin.
The publication of Rocha-Martin, J. et al. (2011), New perspectives of biotechnological NADH oxidase from Thermus thermophilus H827 variant as NAD +-recycling enzyme, BMC Biotechnology 11 (1), 101 describes the immobilization of the enzyme NADH oxidase (NOX) on agarose activated with glyoxyl groups, in which the binding was performed through its regions rich in lysine residues, resulting in strong covalent bonds. This work shows that, depending on the protocol used, the immobilization and the enzymatic activity achieved vary.
Regarding the immobilization of glucuronidase enzymes, the document published by Rapatz, E. et al. (1988), Studies on the immobilization of glucuronidase (Part 2), Applied Biochemistry and Biotechnology, 19 (3), 235-242 indicates that β-glucuronidase is a multimeric enzyme that can dissociate into its subunits, depending on dilution and the pH when being soluble. To prevent dissociation of the enzyme, immobilization of β-glucuronidase from Helix pomatia in bovine serum albumin and crosslinking with glutaraldehyde was tested, which achieved stabilization of the enzyme. Subsequently, U.S. Pat. No. 5,739,004 generally discloses supports for immobilization of enzymes, including β-glucuronidase derived from microorganisms capable of forming spores, such as Candida, Bacillus, Neurospora and Clostridium; where agarose, cellulose and dextran are mentioned, among others.
The state of the art indicates that, depending on the desired application, it is necessary to define a specific immobilization protocol to increase the stability of an enzyme, in which it is necessary to control parameters such as temperature, pH, size and particular characteristics of the polymers used, etc. In particular for analysis of glucuronide metabolites present in biological samples, considering the above background there is a significant need to provide supports for the immobilization of enzymes that increase their stability to facilitate the degradation of these compounds, in order to accelerate the processes of analysis and quantification of drugs and toxins, among others.